The INT3 gene is rearranged by MMTV integration in 20% of virus-induced CzechII strain mouse mammary tumors. In all cases the viral integration is in the same transcription orientation as INT3. One consequence of these integrations is the expression of a truncated INT3 RNA species initiated in the MMTV LTR which encodes the transmembrane and intracellular domains of the normal gene product. We have completed the nucleotide sequence of the normal 6.4 Kb INT3 RNA. It encodes a 200 KD protein which shares 60% homology with the mouse homologue of NOTCH1. We have therefore renamed this gene NOTCH4/INT3. Members of the NOTCH gene family are known to participate in cell fate determination, cellular differentiation, and transcriptional regulation. NOTCH4/INT3 has several novel characteristics which distinguish it from other members of this gene family. The extracellular domain of NOTCH4/INT3 contains 29 instead of 36 EGF-like repeats found in NOTCH1. Four novel EGF-like repeats have been created as consequence of small deletions which have occurred within the gene during evolution. Thus N-terminal end of EGF repeat 14 is fused with a portion of the C-terminus of repeat 15. Similar fusion's have occurred between repeats 16 and 17, repeats 20 and 23, and repeats 31 and 32. From the nucleotide sequence of host- viral junction fragments of 9 independent mammary tumors it was determined that all of the integration events in NOTCH4/INT3 have occurred within a174 bp region 3' of the LIN12 repeat sequences in the extracellular domain and 5' of the transmembrane encoding sequences. This strongly suggests loss of the LIN12 repeat sequences are required for viral induced activation of NOTCH4/INT3. INT6 is a unique gene, highly conserved throughout evolution. It is disrupted by MMTV insertional mutagenesis in mouse mammary tumors and a preneoplastic mammary hyperplastic outgrowth line leading to the expression of a truncated INT6 gene product. Although it is expressed in all adult tissues of the mouse and early in embryonic development, its function is unknown. To study the normal distribution and the potential function of the Int6 gene products, we produced antibodies against synthetic peptides specific for the Int6 protein. Western blot and immunoprecipitation analysis demonstrated a 43 kDa major gene product that is localized in the cytosolic fraction of mammary cell homogenates. This latter observation is supported by immunoperoxidase analysis that shows strong staining anti-Int6 peptide in the perinuclear region of the HC11 mammary epithelial cell line, suggesting a possible localization in the Golgi apparatus. Further immunocytochemical studies in the mouse embryo show that Int6 expression is prevalent in migrating neural crest cells, in the notochord and in condensing cartilage between 9.5 and 14.5 days of development. In these embryonic tissues, Int6 staining colocalizes with the staining of ricinus lectin and giantin, proteins that are specifically associated with the Golgi apparatus. The restricted expression of the protein within the Golgi and its strong conservation throughout evolution suggest that Int6 may perform an essential cellular function. The human homolog of INT6 has been clone the sequence of the encoded protein is identical to the mouse INT6 protein. Human INT6 is located on chromosome 8q22-q23 and is affected by loss of heterozygosity in 28% of primary human breast carcinomas. At the present time no mutations have been found in the remaining allele.